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Vertebrate inner ear develops from its rudiment, otic placode, which later forms otic vesicle and gives rise to tissues comprising the entire inner ear. Although several signaling molecules have been identified as candidates responsible for inner ear specification and patterning, many details remain elusive. Here, we report that Paraxial Protocadherin (PAPC) is required for otic vesicle formation in Xenopus embryos. PAPC is expressed strictly in presumptive otic placode and later in otic vesicle during inner ear morphogenesis. Knockdown of PAPC by dominant-negative PAPC results in the failure of otic vesicle formation and the loss of early inner ear markers Sox9 and Tbx2, suggesting the requirement of PAPC in the early stage of otic vesicle development. However, PAPC alone is not sufficient to induce otic placode formation.
Fig. 1.
Expression profile of PAPC in Xenopus head analyzed by RT-PCR. (A) Total RNA extracted from dissected head of stage 21–45 embryos was reverse transcribed and PCR amplified using PAPC-specific primers. PAPC transcripts were present at each stage of otic placode development. (B) Vertical line indicates the position to dissect heads of stage 21–45 embryos.
Fig. 2.
Spatial localization of Xenopus PAPC transcripts during inner ear development visualized by whole-mount in situ hybridization analysis. Transcripts for PAPC were detected by whole-mount in situ hybridization. (A,C,D) Lateral views are shown with anterior to the left. (B) Dorsal view with anterior to the left. (A) In late neurula (stage 17), bilateral stripes of PAPC expression prefigured the prospective otic placode. PAPC expression was also found in the forming somite. (B) At stage 20, PAPC was expressed in the prospective otic placode. (C) At stage 23, the expression of PAPC was in the otic cup. (D) A transverse section of a stage 23 embryo revealed PAPC transcripts were present in the otic cup. Insertion is the enlargement of (D). (E) At the hatching stage (stage 32), PAPC was clearly expressed in the otic vesicles. (F) A transverse section through the hindbrain region of a stage 32 embryo. PAPC transcripts were detected in the entire otic vesicle. Insertion is the enlargement of (F). Red arrows indicate the otic signal; yellow arrowheads indicate the signals in the posterior region.
Fig. 3.
Reduction of PAPC disrupts the otic vesicle structure. (A,B) Otic vesicle was lost in the embryos when 1.5 ng DN-PAPC and 200 pg β-galactosidase were injected in the right side animal ventralblastomere at the eight-cell stage (n = 40, 85%). (C,D) Otic vesicle was lost in the embryos injected with 400 pg M-PAPC and 200 pg β-galactosidase in the right side animal ventralblastomere at the eight-cell stage (n = 28, 86%). (A,C) Control side. (B,D) Injected side. (B) Pigmented cells aggregated in the injected side (red arrowhead). (D) A few pigmented cells aggregated in the injected area (red arrowhead). Blue color domain is LacZ staining.
Fig. 4.
PAPC is required to specify the otic placode. Expression of Sox9 and Tbx2 in DN-PAPC or M-PAPC injected embryos in stage 32, detected by whole-mount in situ hybridization. (A–F) Sox9 and Tbx2 expression was both lost in embryos injected with 1.5 ng DN-PAPC in the right side animal ventralblastomere at the eight-cell stage. (A,B,C) Sox9 expression (n = 22, 86%). (D,E,F) Tbx2 expression (n = 18, 83%). (G–L) Sox9 and Tbx2 expression was both lost in embryos injected with 400 pg M-PAPC in the right side animal ventralblastomere at the eight-cell stage. (G,H,I) Sox9 expression (n = 15, 86%). (J,K,L) Tbx2 expression (n = 13, 85%). (A,D,G,J) Control side. (B,E,H,K) Injected side. (C,F,I,L) Transverse section through the embryo injected with either 1.5 ng DN-PAPC or 400 pg M-PAPC. Black arrows indicate the positive signal; red arrows indicate the absence of the signal in the injected side.
Fig. 5.
Co-injection of FL-PAPC rescues the otic vesicle defects. Defects of otic vesicle formation and Tbx2 expression are rescued by FL-PAPC co-injected with either DN-PAPC or M-PAPC, detected by whole-mount in situ hybridization in stage 32 embryos. (A,B) Otic vesicle and Tbx2 expression was rescued in embryos co-injected with 1 ng FL-PAPC and 1.5 ng DN-PAPC in the right side animal ventral blastomere at the eight-cell stage (n = 30, 60%). (C,D) Otic vesicle and Tbx2 expression was rescued in embryos co-injected with 1 ng FL-PAPC and 400 pg M-PAPC in the right side animal ventral blastomere at the eight-cell stage (n = 31, 81%). (A,C) Control side. (B,D) Injected side. Black arrows indicate the endogenous positive signal; red arrows indicate the rescued signal in the injected side. (E) Tbx2 expression rescued by co-injection with FL-PAPC. The blue bar depicts the percentage of uninjected sibling embryos; the black bar depicts the percentage of embryos injected with either DN-PAPC or M-PAPC; the dark gray bar depicts the percentage of embryos co-injected of FL-PAPC with either DN-PAPC or M-PAPC. UC, uninjected sibling control embryos; DN, DN-PAPC; M, M-PAPC; FL, FL-PAPC.
Fig. 6.
PAPC is not sufficient to initiate formation of the otic placode. Embryos injected with FL-PAPC (800 pg) and β-galactosidase (200 pg) mRNA into one of two blastomeres at two-cell stage, fixed, and stained for expression of Sox9 (A) and Tbx2 (B) at stage 32. (A) Microinjection of FL-PAPC did not induce ectopic Sox9 expression (n = 14, 100%). (B) Microinjection of FL-PAPC did not induce ectopic Tbx2 expression (n = 12, 100%). Black arrows indicate the endogenous positive signal.
